The present invention relates to novel polyamide conjugates, processes for their preparation and the use of these conjugates in therapeutic compositions.
The increased shortage of donor organs for transplantation from human to human has led to a great interest in the possibilities of xenotransplantation (transplantation from non-human to human). At present research is focused on easily accessible animals, such as pigs, as the source for organs. However, pig to human xenotransplantation has to over-come a series of obstacles the most immediate and striking of them being hyperacute rejection (HAR). HAR is caused by pre-formed xe2x80x98naturalxe2x80x99 polyclonal antibodies which are mostly directed against carbohydrate cell surface epitopes containing terminal Gal xcex11,3Gal structures (anti-xcex1Gal). In addition, other antibodies may also exist directed against N-glycolyl neuraminic acid structures also present on pig endothelium.
To achieve long term graft survival, strategies which address the xenoreactive antibodies are needed. One approach is removal of antibodies by injection of high affinity ligands which may act as inhibitors of antibody deposition on transplanted tissue. Another approach is immunoapheresis, a further development of plasmapheresis, a clinically established procedure similar to dialysis. Immunoapheresis involves the extracorporal treatment of blood by first separating the blood cells, then passage of plasma through immunoaffinity columns, re-mixing of blood cells with the antibody depleted plasma and reintroducing the blood into the patient.
Thus, there is a need for ligands able to bind intracorporally and for a column material able to bind extracorporally the polyclonal xenoreactive antibodies with improved affinity.
The present invention relates to a polyamide conjugate comprising
either (a)a xenoantigenic group;
or (b)a biologically active group and a macromolecular, macro- or microscopic entity; bound to a polyamide backbone
wherein the polyamide backbone comprises at least one structural element of formula I 
and in case (b) additionally at least one structural element of formula II 
in which
each of A and Axe2x80x2, independently, is a trivalent bridging group;
each of R1 and R1xe2x80x2, independently, is a direct bond or C1-C6alkylene;
each of X1 and X1xe2x80x2, independently, is xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)NRxe2x80x94, xe2x80x94NRxe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94Oxe2x80x94;
each of R2 and R2xe2x80x2, independently, is a direct bond or a bivalent bridging group;
each of X2 and X2xe2x80x2, independently, is a direct bond or xe2x80x94Oxe2x80x94 or xe2x80x94NRxe2x80x94; wherein R is hydrogen, OH, C1-C12alkyl, C2-C12alkenyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C2-C7heterocycloalkyl, C2-C11heterocycloalkenyl, C6- or C10aryl, C5-C9heteroaryl, C7-C16aralkyl, C8-C16aralkenyl with C2-C6alkenylene and C6- or C10aryl, or di-C6- or C10aryl-C1-C6alkyl; and
each of Y and Yxe2x80x2, independently, is a direct bond or a bivalent bridging group; with the proviso that X1 or X1xe2x80x2 is not xe2x80x94NRxe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94Oxe2x80x94 when R1 or R1xe2x80x2 is a direct bond.
When the polyamide backbone of the invention comprises more than one structural element of formula I or II, these elements may be identical or different. It may be homopolymeric or copolymeric, or copolymeric additionally having comonomer units of e.g. other aminocarboxylic acids. The copolymeric backbones may be block or statistical polymers. When the trivalent bridging group is chiral, the polyamide backbones may homogeneously have the L- or D-configuration or contain structural elements of both configurations, as homogeneous blocks or as statistical mixtures including racemates (1:1 mixtures).
The average sum of structural elements of the polyamide backbone [n] may be in the range of from 10 to 10,000, preferably of from 50 to 1,500, more preferably of from 250 to 1,200, most preferably of from 900 to 1200. Polydispersity may range from 1.001 to 2.0, preferably from 1.1 to 1.5, more preferably from 1.15 to 1.2.
Any alkyl and alkylene radical or moiety may be linear or branched. Preferably alkyl is C1-C18alkyl, more preferably C1-C4alkyl, and may be e.g. methyl, ethyl, n- or i-propyl, or n-, i- or t-butyl. Preferably alkenyl may contain 2 to 7 C atoms. Aryl or heteroaryl may be a 5 or 6 membered ring or a bicyclic radical of two fused rings, one or more heteroatoms chosen from the group Oxe2x80x94, Nxe2x80x94 and S-atom being present in the heteroaryl. Examples include phenyl, naphthyl, furanyl, pyrrolyl, etc. Aralkyl preferably has 7 to 12 C atoms and may be phenyl-C1-C6alkyl, e.g. benzyl or phenethyl. An example for aralkenyl is cinnamyl.
A or Axe2x80x2 may be a single atom with at least three valences, e.g. C, N or Si; in particular 
wherein Raa is H, C1-C6alkyl, C2-C6alkenyl, C3-C7cycloalkyl, phenyl or benzyl.
R2 or R2xe2x80x2 as a bivalent bridging group may contain from 1 to 35, preferably from 1 to 20, particularly from 1 to 16 C atoms, e.g. C1-C35alkylene, C2-C8alkenylene, C2-C8alkynylene, C3-C12cycloalkylene, C6-C10arylene or C7-C35aralkylene wherein the alkylene, alkenylene and alkynylene radicals or moieties may be interrupted by one or more groups selected from xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94SO2xe2x80x94 and xe2x80x94HNxe2x80x94.
The bridging group R2 or R2xe2x80x2 may, for example, conform to formula III
xe2x80x94R5xe2x80x94X5xe2x80x94R6xe2x80x94X4xe2x80x94R7xe2x80x94xe2x80x83xe2x80x83(III)
in which
each of X5 and X4, independently, is a direct bond, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94OC(O)Oxe2x80x94, xe2x80x94SO2Oxe2x80x94, xe2x80x94OSO2xe2x80x94, xe2x80x94OSO2Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94C(O)NHxe2x80x94, xe2x80x94NHC(O)Oxe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94NHC(O)NHxe2x80x94, xe2x80x94NHSO2xe2x80x94, xe2x80x94SO2NHxe2x80x94, xe2x80x94NHSO2Oxe2x80x94, xe2x80x94OSO2NHxe2x80x94 or xe2x80x94NHSO2NHxe2x80x94;
each of R5 and R7, independently, is a direct bond, C1-C20alkylene, C5- or C6-cycloalkylene, C6-C10arylene or C7-C12aralkylene; and
R6 is a direct bond or C1-C20alkylene which is optionally interrupted by one or more, preferably 2 O atoms;
with the proviso that when R6 is a direct bond X4 is also a direct bond.
Preferably R2 or R2xe2x80x2 may be oxyalkylene or polyoxyalkylene, more preferably having from 2 to 4 C atoms, particularly 2 or 3 C atoms, in the alkylene and from 2 to 20, preferably from 2 to 10, alkylene units, especially oxypropylene and polyoxypropylene, e.g. polyoxypropylene having from 2 to 20 preferably from 2 to 10, oxypropylene units.
A polyamide conjugate comprising a xenoantigenic group and no macromolecular, macro- or microscopic entity will hereinafter be referred to as xe2x80x9cconjugate Type Ixe2x80x9d and a polyamide conjugate comprising a biologically active group and a macromolecular, macro- or micro-scopic entity will hereinafter be referred to as xe2x80x9cconjugate Type IIxe2x80x9d.
In conjugates Type I the xenoantigenic group is conjugated to the polyamide backbone via Y of a structural element of formula I. The conjugates Type I may comprise one or more identical or different xenoantigenic groups.
A xenoantigenic group may be any group identifiable by known methods, e.g. as disclosed in U.S. Pat. No. 5,695,759 (the contents thereof with respect to the method being incorporated herein by reference), comprising isolating xenoantibodies from human blood by perfusing the blood over xenograft material, e.g. removing bound antibodies from the xenograft and using those antibodies to screen candidate epitopes, e.g. by a suitable immunoassay.
The xenoantigenic group may preferably be derived from an oligosaccharide terminating with an xcex1-linked D-galactopyranose or N-glycoyl-neuraminic acid at its reducing end.
Typically (and with respect to conjugates Type I and II) the oligosaccharide may consist of from 1 to 20, preferably 1 to 15, particularly 1 to 10 sugar monomers selected from naturally occurring and modified sugar monomers. The skilled person is familiar with naturally occurring and modified sugar monomers and oligosaccarides comprising these sugar monomers from the standard works of organic chemistry or biochemistry, for example the Specialist Periodical Reports edited at the beginning by The Chemical Society and now by The Royal Society of Chemistry London, e.g. Ferrier et al. Carbohydrate Chemistry 29 The Royal Society of Chemistry London (1997).
Examples of sugar monomers include D- and L-aldopyranoses and D- and L-aldofuranoses, for example glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose and talose, D- and L-ketopyranoses and D- and L-ketofuranoses, for example dihydroxyacetone, erythrulose, ribulose, xylulose, psicose, fructose, sorbose and tagatose, and also D- and L-diketopyranoses, for example pentodiulose and hexodiulose.
The term sugar monomers also includes those sugar monomers which are modifications of the examples listed, for example, protected, partially protected or unprotected deoxysugars of the D- and L-configurations, preferably 2-, 3-, 4-, 5- and 6-deoxyaldoses, such as fucose, rhamnose and digitoxose, 1,2-dideoxyaldoses, such as glucal, galactal and fucal, and 1-, 3-, 4-, 5- and 6-deoxyketoses, 2-, 3-, 4-, 5- and 6-deoxyaminosugars of the D and L configurations, such as glucosamine, mannosamine, galactosamine and fucosamine, and deoxyacylaminosugars, such as N-acylglucosamine, N-acylmannosamine, N-acylgalactosamine and N-acyl-fucosamine, preferably their C1-C4alkyl esters. In addition, these modifications are understood to mean aldonic, aldaric and uronic acids, such as gluconic acid or glucuronic acid, and also ascorbic acid and amino acid-carrying sugar monomers. Modified sugar monomers are also understood to mean those having a carbon chain which is longer than 6 C atoms, such as heptoses, octoses, nonoses, heptuloses, octuloses and nonuloses, and also their representatives which are substituted in accordance with the above-listed criteria, such as ketodeoxyoctanic acid, ketodeoxynonanic acid, N-acylneuraminic acids and N-acylmuraminic acids.
If two, three, four or five of the abovementioned, identical or different monomers are assembled the resulting saccharides are denoted as di-, tri-, tetra- or pentasaccharides. The linkage is preferably xcex1-O-glycosidic or xcex2-O-glycosidic, e.g. (1xe2x86x922)-, (1xe2x86x923)-, (1xe2x86x924)-, (1xe2x86x925)-, (1xe2x86x926)-, (2xe2x86x923)- and (2xe2x86x926)-glycosidic linkages. Examples of disaccharides are e.g. trehalose, sophorose, kojibiose, laminaribiose, maltose, cellobiose, isomaltose, gentibiose, sucrose and lactose, and their derivatives. Examples of trisaccharides are raffinose and melezitose. Furthermore, the oligosaccharides may be linked via S-, N- and C-glycosidic linkages, e.g. xe2x80x94Sxe2x80x94, xe2x80x94NR12xe2x80x94, xe2x80x94NR12C(O)xe2x80x94 or xe2x80x94CR13R14xe2x80x94, wherein R12, R13 and R14 independently of each other are H, C1-C12alkyl, C5- or C6cycloalkyl, C5- or C6cycloalkylmethyl or -ethyl, phenyl, benzyl or phenethyl.
In conjugates Type I the following significances are preferred either individually or in any combination or sub-combination:
(a)A is C1-C6alkanetriyl, more preferably methanetriyl.
(b)R1 is C1-C6alkylene, more preferably xe2x80x94(CH2)4xe2x80x94;
(c)X1 is xe2x80x94NRxe2x80x94 wherein R has the meanings mentioned above, more preferably X1 is xe2x80x94NHxe2x80x94;
(d)R2 is a direct bond;
(e)X2 is a direct bond;
(f)Y is a group of formula III wherein R5 is C1-C8alkylene, X5 is xe2x80x94NHxe2x80x94, R6 is a direct bond, X4 is xe2x80x94C(O)xe2x80x94, and R7 is C1-C8alkylene,
preferably Y is a group of formula IIIa
xe2x80x94(CH2)mNHC(O)(CH2)3xe2x80x94xe2x80x83xe2x80x83(IIIa)
wherein m is a number from 1 to 8, preferably 1 to 6; and
wherein with respect to formula I xe2x80x94(CH2)3xe2x80x94 is bound to S;
(g)the xenoantigenic group, hereinafter referred to as R3a, is a group of formula IVa1, IVb1, IVc1, IVd1 or IVe1
xe2x80x83wherein the individual Rz are independently ORz1, SRz1 or NHXzRz1 wherein Xz is xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94S(O)2xe2x80x94, xe2x80x94C(O)Yxe2x80x94 or xe2x80x94C(S)Yxe2x80x94, in which Y is xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94C1-C6alklene, xe2x80x94NHxe2x80x94C1-C6alkylene, xe2x80x94Oxe2x80x94C1-C6alkylene, xe2x80x94C1-C6alkylene-Oxe2x80x94, xe2x80x94C1-C6salkylene-Sxe2x80x94, xe2x80x94C1-C5alkylene-NHxe2x80x94, xe2x80x94C1-C6alkylene-Oxe2x80x94C(O)Oxe2x80x94, xe2x80x94C1-C6alkylene-Sxe2x80x94C(O)Oxe2x80x94 or xe2x80x94C1-C6alkylene-NHxe2x80x94C(O)Oxe2x80x94 and Rz1 is hydrogen, C1-C20alkyl, C2-C20alkenyl, C3-C15cycloalkyl, C3-C15cycloalkenyl, C6-C10aryl, C7-C20aralkyl or C2-C9heteroaryl, where alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, aralkyl and heteroaryl are unsubstituted or substituted by one or more substituents selected from OH, halogen, halo-C1-C20alkyl, nitro, C1-C18alkyl, C1-C8alkoxy, C1-C8alkenyloxy, amino, mono-C1-C18alkylamino, di-C1-C18alkylamino, benzylamino, SO3H, SH, thio-C1-C18alkyl and NHC(O)xe2x80x94C1-C18alkyl; and
Zz is xe2x80x94Oxe2x80x94 or xe2x80x94NHC(O)xe2x80x94;
preferably a group of formula IVa, IVb, IVc, IVd, IVe or IVf 
Particularly preferred are conjugates Type I wherein R3axe2x80x94Yxe2x80x94 is a group of formula Va, Vb, Vc, Vd, Ve or Vf 
A preferred subgroup are conjugates Type I comprising at least one structural element of formula I* 
wherein
A, R1, X1, R2, X2 and Y are as defined above and R3a is a xenoantigenic group, more preferred a structural element of formula Ia1a, most preferred a structural element of formula Iaa, 
xe2x80x83in which Y is a group of formula IIIa and R03a is a group of formula IVa, IVb, IVc, IVd, IVe or IVf.
Particularly preferred are conjugates Type I comprising at least one structural element of formula Iaa wherein xe2x80x94Yxe2x80x94R03a is a group of formula Va, Vb, Vc, Vd, Ve or Vf.
According to the invention the polyamide backbone of conjugates Type I may additionally comprise at least one structural element of formula VI, 
in which
Axe2x80x3, R1xe2x80x3, X1xe2x80x3, R2xe2x80x3 and X2xe2x80x3 independently have the meanings and preferences as defined for A, R1, X1, R2 and X2, respectively, above and
R8 is a polar group, preferably polyhydroxy-C2-C12alkyl or xe2x80x94C3-C12cycloalkyl, more preferably polyhydroxy-C2-C6alkyl or xe2x80x94C3-C6cycloalkyl, with 1 to 6, preferably 2 to 5 hydroxy groups, most preferably xe2x80x94CH2CHOHCH2OH.
A preferred subgroup of structural elements of formula VI are those of formula VIa 
wherein R8 is as defined above.
Particularly preferred structural elements of formula VI are those of formula VIb1
more preferred those of formula VIb 
Particularly preferred are conjugates Type I comprising at least one structural element of formula I* and at least one structural element of formula VI wherein A, Axe2x80x3, R1, R1xe2x80x3, X1, X1xe2x80x3, R2, R2xe2x80x3, X2, X2xe2x80x3, Y, R3a and R8 have the meanings and preferences as mentioned above.
Most preferred are conjugates Type I comprising at least one structural element of formula Iaa wherein xe2x80x94Yxe2x80x94R03a is a group of formula Va, Vb, Vc, Vd, Ve or Vf and at least one structural element of formula VIb.
In the novel conjugates Type I the content of the structural elements of formulae I* and VI may for example be from 0.1 to 99.9 mol %, the values adding up to 100% in the case of conjugates Type I only consisting of structural elements of formula I* and VI. In the case of structural elements of formula I* the content may for example be from 1 to 50%, preferably from 10 to 30%. In the case of structural elements of formula VI the content may be from 0 to 99%, preferably from 70 to 90%.
Preferred conjugates Type I are those wherein the average sum of structural elements [n] is in the range of from 200 to 300 with a polydispersity of from 1.1 to 1.2 and the ratio of structural elements of formula Iaa [x] is 5 to 30% or those wherein [n] is in the range of from 900 to 1200 with a polydispersity of from 1.1 to 1.2 and [x] is 5 to 50%; with a ratio of structural elements of formula VIb [z] ranging from 45 to 94%; R03axe2x80x94Yxe2x80x94 being identical or different and selected from groups of formula Va, Vb, Vc, Vd, Ve and Vf.
Examples of preferred conjugates Type I are those wherein
(a) n is 250, [x] is 25% when R3axe2x80x94Yxe2x80x94 is a group of formula Vb; +75% [z];
(b) n is 250, [x] is 8% when R3axe2x80x94Yxe2x80x94 is a group of formula Vc; +92% [z];
(c) n is 250, [x] is 16% when R3axe2x80x94Yxe2x80x94 is a group of formula Vc; +84% [z];
(d) n is 250, [x] is 41% when R3axe2x80x94Yxe2x80x94 is a group of formula Vc; +59% [z];
(e) n is 250, [x] is 61% when R3axe2x80x94Yxe2x80x94 is a group of formula Vc; +39% [z];
(f) n is 250, [x] is 23% when R3axe2x80x94Yxe2x80x94 is a group of formula Vd; +77% [z];
(g) n is 250, [x] is 22% when R3axe2x80x94Yxe2x80x94 is a group of formula Vf; +78% [z];
(h) n is 1000, [x] is 24% when R3axe2x80x94Yxe2x80x94 is a group of formula Vc; +76% [z];
(i) n is 1050, [x] is 21% when R3axe2x80x94Yxe2x80x94 is a group of formula Vb; +79% [z];
(j) n is 1050, [x] is 28% when R3axe2x80x94Yxe2x80x94 is a group of formula Vc; +72% [z];
(k) n is 1050, [x] is 25% when R3axe2x80x94Yxe2x80x94 is a group of formula Vf; +75% [z]; and
(l) n is 1050, [x] is 27% when R3axe2x80x94Yxe2x80x94 in a third of structural elements of formula I* is a group of formula Vb, in another third is a group of formula Vc and in another third is a group of formula Vf; +73% [z];
particularly examples (i) to (l).
The novel conjugates Type I are obtainable by a process comprising reacting a polyamide comprising at least one structural element of formula VII 
in which A, R1, X1, R2 and X2 are as defined above and X3 is halogen, obtainable e.g. as disclosed in WO 97/19105, with a thiol of formula VIIIaxe2x80x2
R3axe2x80x94Yxe2x80x94SHxe2x80x83xe2x80x83(VIIIaxe2x80x2),
in which R3a and Y are as defined above.
The thiols are either known or may be prepared in accordance with known methods, e.g. by reacting an amino-functionalized xenoantigen, e.g. the oligosaccharide, with a thiolactone.
When the xenoantigen is a saccharide it may be prepared according to conventional chemical procedures, using enzymes or by following a mixed approach. The use of enzymes for the preparation of oligosaccharide derivatives is disclosed e.g. in WO 97/28173 and WO 97/128174.
Conjugates Type I additionally comprising at least one structural element of formula VI are obtainable by a process comprising reacting a polyamide comprising at least two structural elements of formula VII, with one or more compounds selected from the group of a thiol of formula Villaxe2x80x2 and a thiol of formula IX
R8xe2x80x94SHxe2x80x83xe2x80x83(IX)
in which R8 is as defined above.
Preferably the reaction may be carried out in the presence of a strong, non-nucleophilic base, preferably an organic base having at least one tertiary N atom; particularly bicyclic or polycyclic amines. Examples are quinuclidine and 1,8-diazabicyclo[5.4.0]undec-7-ene(1,5-5) (DBU). The base may be employed in at least equimolar quantities, preferably in slight excesses.
The reaction may also be carried out using alkali metal thiolates R3aSM wherein M is an alkali metal, for example Li or Na.
A polar, aprotic solvent, preferably a nitrogen-dialkylated carboxyimide, lactam, sulfoxide or sulfone, for example dimethylformamide, may be used. The reaction may be effected either without or with the addition of water, for example up to quantities at which the polymer remains in solution. Non-limiting details for the preparation of the conjugates Type I are disclosed in Examples A through C1.
The novel conjugates Type I have interesting properties. In particular they have affinity for human polyclonal xenoreactive antibodies present in body fluids, e.g. blood, and are useful as ligands or depleting agents. Preferably the conjugates Type I are used for intracorporal depletion of xenoantibodies, comprising administering, e.g. by injection, infusion or perfusion, into a xenograft recipient prior to and/or after transplantation of the xenograft a conjugate Type I. The dosage may be from 0.0001 to 10, preferably from 0.01 to 5, more preferably from 0.2 to 2 mg per kg bodyweight per injection. The administration may be repeated as required prior to transplantation and/or after transplantation as long as removal of antibodies is of therapeutic benefit.
The novel conjugates Type I may also be used as a pharmaceutical for inducing tolerance or anergy towards the xenoantigenic epitopes or to specifically target B cells with xenoantigen receptors. When administered, e.g. by injection, into a xenograft recipient prior to transplantation of the xenograft the dosage of a conjugate Type I may be from 0.0001 to 10, preferably from 0.001 to 5, more preferably from 0.02 to 2 mg per kg bodyweight per injection. The administration may be repeated as required to achieve tolerance.
The conjugates Type I may be administered as a single conjugate or as a mixture of different conjugates Type I as such or in the form of a composition adapted for intravenous administration, e.g. together with one or more pharmaceutically acceptable carrier or diluent therefor. Such mixture may comprise conjugates Type I differing with respect to the polyamide backbone and/or to xenoantigenic group.
Preferred are compositions wherein the conjugate Type I comprises identical or different xenoantigenic groups or compositions comprising a mixture of conjugates Type I, each conjugate comprising identical or different xenoantigenic groups, the xenoantigenic groups being derived from a disaccharide, preferably a group of formula IVb, from a trisaccharide, preferably a group of formula IVc or IVd, or from a pentasaccharide, preferably a group of formula IVe or IVf. Preferably the composition may comprise a conjugate Type I comprising a group derived from a disaccharide, a group derived from a trisaccharide and a group derived from a pentasaccharide, in a ratio of from 5 to 0.1:5 to 0.1:5 to 0.1, preferably in a ratio of 1:1:1 or a mixture of conjugates Type I each conjugate comprising identical xenoantigenic groups, the xenoantigenic groups being derived from a disaccharide, a trisaccharide or a pentasaccharide, the conjugates being present in the composition in a ratio of from 5 to 0.1:5 to 0.1:5 to 0.1, preferably in a ratio of 1:1:1.
In accordance with the foregoing the present invention further provides:
(a) a conjugate Type I for use as a pharmaceutical, preferably as an agent for removing xenoantibodies from human body fluid or as a ligand to present xeno-antigen in the induction of tolerance or anergy;
(b) a method for removing xenoantigenic antibodies from a xenograft recipient, which method comprises intracorporally contacting said body fluid with a conjugate Type I as under (a);
(c) a method for inducing tolerance or anergy towards the xenoantigenic epitopes or to specifically target B cells with xenoantigen receptors in a subject in need of such treatment, which method comprises administering, e.g. by injection, infusion or perfusion, into a xenograft recipient prior to and/or after transplantation of the xenograft an effective amount of a conjugate Type I as under (a);
(d) a composition comprising a conjugate Type I as under (a) or a mixture of such conjugates Type I as hereinbefore disclosed for use preferably in a method for removing xenoantibodies from human body fluid or for inducing tolerance or anergy towards the xenoantigenic epitopes or to specifically target B cells with xenoantigen receptors.
In conjugates Type II the biologically active group is conjugated to the polyamide backbone via Y of a structural element of formula I and the macromolecular, macro- or microscopic entity is conjugated to the polyamide backbone via Yxe2x80x2 of a structural element of formula II. The conjugates Type II may comprise one or more identical or different biologically active groups. The conjugates Type II may comprise one or more identical or different macromolecular, macro- or microscopic entities. A single entity may be bound to one or simultaneously to 2 or more structural elements from the same polyamide backbone. Alternatively a single entity may simultaneously be bound to one or more structural elements from different polyamide backbones.
Examples for a biologically active group are radicals derived from alkaloids, carbohydrates, vitamins, peptides, proteins, conjugated proteins, lipids, terpenes, oligonucleotides, antigens and antibodies; or any other ligand, which is preferably recognised in a multivalent form by its receptor or a cellular surface. Preferably the biologically active group is derived from an antigen, preferably from a xenoantigen, more preferred from an oligosaccharide terminating with an xcex1-linked D-galactopyranose or N-glycoyl-neuraminic acid at the reducing end, e.g. as hereinbefore described for the conjugates Type I.
Examples for macromolecular, micro- and macroscopic entities are globular proteins such as serum albumin or KLH; polymers such as poly-amides, poly-imides, poly-ethylene glycols, poly-styrenes, poly-acrylates, poly-acrylamides, co- and block-polymers thereof, natural and modified polysaccharides; glass beads, silica gel, diatomeous earth and structures formed from aggregated or crosslinked lipid mono- or bi-layers, or multiple bilayers. If the macromolecular, micro- or macroscopic entity is a polysaccharide it may consist of more than 20 sugar monomers with a molecular weight of from a few thousand to as high as 100 million. The skilled person is familiar with natural and modified polysaccharides which differ in the nature of their recurring monosaccharide units, in the length of their chains, and in the degree of branching. Modified natural polysaccharides may comprise derivatives obtainable by reacting natural polysaccharides with mono-, bi- or polyfunctional reagents or oxidants, which may substantially be based on modifications of the hydroxy groups of the polysaccharides by ether or ester forming reactions or selective oxidations. Particularly useful are cross-linked, preferably three-dimensionally linked polysaccharides. Examples for natural and modified polysaccharides are natural and modified starches, celluloses, dextrans and agaroses. Particularly useful as cross-linked, preferably three-dimensionally linked polysaccharides are e.g. Sephacryl(copyright) and Sepharose(copyright) and their derivatives, e.g. NHS-activated CH-Sepharose 4B or NHS-activated Sepharose 4 Fast Flow (commercially available).
In conjugates Type II each of A, Axe2x80x2, R1, R1xe2x80x2, X1, X1xe2x80x2, R2, R2xe2x80x2, X2 and X2xe2x80x2, independently, has one of the preferred significances given above for the conjugates Type I either individually or in any combination or sub-combination, including:
(g) Yxe2x80x2 is a group of formula III wherein R5 is a direct bond, X5 is xe2x80x94NHxe2x80x94, R6 is xe2x80x94[(CH2)2O]2(CH2)2xe2x80x94, X4 is xe2x80x94NHC(O)xe2x80x94, and R7 is C1-C8alkylene,
preferably Yxe2x80x2 is a group of formula IIIb
xe2x80x94NH[(CH2)2O]2(CH2)2NHC(O)(CH2)3xe2x80x94xe2x80x83xe2x80x83(IIIb)
wherein with respect to formula II xe2x80x94(CH2)3xe2x80x94 is bound to S;
(h) the biologically active group, hereinafter referred to as R3, is a group of formula IVa1, IVb1, IVc1, IVd1 or IVe1 as above, preferably a group of formula IVa, IVb, IVc, IVd, IVe or IVf as above;
(i) the macromolecular, macro- or microscopic entity, hereinafter referred to as R4, is preferably derived from a natural or modified polysaccharide, more preferably from cellulose, agarose or sepharose, particularly from NHS-activated CH-Sepharose 4B or NHS-activated Sepharose 4 Fast Flow, most preferably from NHS-activated Sepharose 4 Fast Flow.
Particularly preferred are conjugates Type II wherein R3xe2x80x94Yxe2x80x94 is a group of formula Va, Vb, Vc, Vd, Ve or Vf as above.
A preferred subgroup are conjugates Type II comprising at least one structural element of formula I** 
wherein
A, R1, X1, R2, X2 and Y are as defined above and R3 is a biologically active group, more preferred a structural element of formula Ia1, 
xe2x80x83most preferred a structural element of formula Ia, 
xe2x80x83in which Y is a group of formula IIIa and R03 is a group of formula IVa, IVb, IVc, IVd, IVe or IVf.
Particularly preferred are conjugates Type II comprising at least one structural element of formula Ia wherein xe2x80x94Yxe2x80x94R03 is a group of formula Va, Vb, Vc, Vd, Ve or Vf.
Particularly preferred are conjugates Type II wherein R4xe2x80x94Yxe2x80x2xe2x80x94 is a group of formula Vg 
A preferred subgroup are conjugates Type II comprising at least one structural element of formula II* 
wherein
A, R1, X1, R2, X2 and Y are as defined above and R4 is a macromolecular, macro- or microscopic entity,
more preferred a structural element of formula IIa1,
xe2x80x83most preferred a structural element of formula IIa, 
xe2x80x83in which xe2x80x94Yxe2x80x2xe2x80x94R04 is a group of formula Vg.
According to the invention the polyamide backbone of conjugates Type II may additionally comprise at least one structural element of formula VI with all the meanings and preferences as above.
Particularly preferred are conjugates Type II comprising at least one structural element of formula I**, at least one structural element of formula II* and at least one structural element of formula VI wherein A, Axe2x80x2, Axe2x80x3, R1, R1xe2x80x2, R1xe2x80x3, X1, X1xe2x80x2, X1xe2x80x3, R2, R2xe2x80x2, R2xe2x80x3, X2, X2xe2x80x2, X2xe2x80x3, Y, Yxe2x80x2, R3, R4 and R8 have the meanings and preferences as mentioned above.
Most preferred are conjugates Type II comprising at least one structural element of formula Ia wherein xe2x80x94Yxe2x80x94R03 is a group of formula Va, Vb, Vc, Vd, Ve or Vf, at least one structural element of formula IIa and at least one structural element of formula VIb.
In the novel conjugates Type II the content of the structural elements of formulae I**, II* and VI may for example be from 0.1 to 99.9 mol %, the values adding up to 100% in the case of conjugates Type II only consisting of structural elements of formulae I**, II* and VI. In the case of structural elements of formula I** the content may for example be from 1 to 50%, preferably from 10 to 30%. In the case of structural elements of formula II* the content may for example be from 0.1 to 20%, preferably from 0.1 to 5%. In the case of structural elements of formula VI the content may be from 0 to 98.9%, preferably from 50 to 98.9%. Examples for useful ratios are disclosed in the following table:
Preferred conjugates Type II are those wherein the average sum of structural elements [n] is in the range of from 200 to 300 with a polydispersity of from 1.1 to 1.2, the ratio of structural elements of formula Ia [x] wherein R03xe2x80x94Yxe2x80x94 is a group selected from a group consisting of groups of formula Va, Vb, Vc, Vd, Ve and Vf is 5 to 30% and the ratio of structural elements of formula IIb (intermediate to structural element of formula IIa, see below) [y]
wherein Zxe2x80x94Yxe2x80x3 is H2N[(CH2)2O](CH2)2NHC(O)(CH2)3xe2x80x94 is from 1 to 5%;
or those wherein [n] is in the range of from 900 to 1200 with a polydispersity of from 1.1 to 1.2, [x] is 5 to 50% and [y] is from 1 to 5%;
with a ratio of structural elements of formula VIb [z] ranging from 45 to 94%.
Examples of preferred conjugates Type II are those wherein
(a) n is 250, x is 25% when R3xe2x80x94Yxe2x80x94 is a group of formula Vc and y is 2%; +73% [z];
(b) n is 250, x is 14% when R3xe2x80x94Yxe2x80x94 is a group of formula Vc and y is 2.6%; +83.4% [z];
(c) n is 250, x is 21.5% when R3xe2x80x94Yxe2x80x94 is a group of formula Va and y is 2%; +76.5% [z];
(d) n is 1050, x is 13% when R3xe2x80x94Yxe2x80x94 is a group of formula Vc and y is 2.6%; +84.4% [z];
(e) n is 1050, x is 16% when R3xe2x80x94Yxe2x80x94 is a group of formula Vb and y is 2.5%; +81.5% [z];
(f) n is 1050, x is 12.5% when R3xe2x80x94Yxe2x80x94 is a group of formula Vf and y is 2.5%; +85% [z]; or
(g) n is 1050, x is 16% when R3xe2x80x94Yxe2x80x94 is a group of formula Ve and y is 2.5%; plus 81.5% [z];
particularly examples (d) to (g).
The novel conjugates Type II are obtainable by a process comprising reacting a polyamide comprising at least two structural elements of formula VII as above with one or more compounds or entities selected from the group of a thiol of formula VIII
R3xe2x80x94Yxe2x80x94SHxe2x80x83xe2x80x83(VIII),
in which R3 and Y are as defined above,
a thiol of formula VIIIa
Zxe2x80x94Yxe2x80x3xe2x80x94SHxe2x80x83xe2x80x83(VIIIa)
wherein Yxe2x80x3 is a bridging group of formula III wherein R5 is a direct bond and the other meanings are as defined above, and Z is hydrogen or a protecting group for X4, preferably an amino protecting group,
and an appropriately derivatized macromolecular, macro- or microscopic entity.
Polyamide conjugates comprising a structural element resulting from the reaction of a structural element of formula VII with a thiol of formula VIIIa may, after elimination of the protecting group Z, conveniently be further reacted with a thiol of formula VIII or an appropriately derivatized macromolecular, macro- or microscopic entity.
The term xe2x80x9cappropriately derivatized macromolecular, macro- or microscopic entityxe2x80x9d is to be understood as denoting a macromolecular, macro- or microscopic entity carrying at least one functional group suitable to react with a structural element of formula IIb. Examples for functional groups useful for coupling of large entities are known to the skilled artisan and described e.g. in Gabius and Gabius (Eds.) Lectins and Cancer 53ff, Springer Verlag, Berlin Heidelberg (1991). Examples are amine/activated carboxylate (N-hydroxy-succinimidyl ester, pentafluorophenyl ester), amine/epoxide, amine/isocyanate, amine/isothiocyanate, amine Michael-acceptor (maleimide), thiol/thiophil and amine/aldehyde.
The derivatized macromolecular, macro- or microscopic entity may be prepared by reacting a macromolecular, macro- or microscopic entity with known bifunctional linkers.
The thiols of formulae VIII and VIIIa may be obtained as disclosed above for the thiols of formula VIIIaxe2x80x2.
Conjugates Type II additionally comprising a structural element of formula VI are obtainable by a process comprising reacting a polyamide comprising at least three structural elements of formula VII, with one or more compounds or entities selected from the thiols of formulae VIII and VIIIa, an appropriately derivatized macromolecular, macro- or microscopic entity and a thiol of formula IX as above. In particular the process may comprise reacting a polyamide comprising at least three structural elements of formula VII, with one or more compounds or entities selected from the thiols of formulae VIII, VIIIa and IX as above, eliminating the protecting group Z and further reacting the resulting compound with an appropriately derivatized macromolecular, macro- or microscopic entity.
Reaction conditions may be in accordance with the reaction conditions as described for conjugates Type I above.
Non-limiting details for the preparation of the conjugates Type II are disclosed in Examples A through D.
The novel conjugates Type II have interesting properties. In particular they have affinity for human polyclonal xenoreactive antibodies and are useful as adsorbent in the affinity chromatography of body fluids. Preferably the body fluid is blood, particularly blood plasma.
Preferably the conjugates Type II are used for extracorporal removal of antibodies from a body fluid, comprising withdrawing antibody-containing body fluid from a xenograft recipient prior to and/or after transplantation of the xenograft, removing preformed antibodies from the fluid via affinity chromatography comprising the conjugates Type II as adsorbent, and reintroducing the antibody-depleted body fluid into the recipient.
The affinity chromatography may preferably be performed as column chromatography wherein the column is packed with conjugate Type II in a manner known to those skilled in the art and described e.g. in U.S. Pat. No. 5,149,425, the contents thereof with respect to filling, storage and use of such columns being incorporated by reference.
The conjugates Type II may be used as a single conjugate or as a mixture of different conjugates Type II as such or in the form of a composition adapted for affinity chromatography. Such mixture may comprise conjugates Type II differing with respect to polyamide backbone, macromolecular, macro- or microscopic entity and/or to antigenic group.
Preferred are compositions wherein the conjugate Type II comprises identical or different xenoantigenic groups or compositions comprising a mixture of conjugates Type II each conjugate comprising identical or different xenoantigenic groups, the xenoantigenic groups being derived from a disaccharide, preferably a group of formula IVb, from a trisaccharide, preferably a group of formula IVc or IVd, or from a pentasaccharide, preferably a group of formula IVe or IVf.
The method for removing the antibodies from the patient""s body fluid may be performed according to known methods, e.g. as disclosed in U.S. Pat. No. 5,695,759. Preferred contact temperatures may be in the range of from 5xc2x0 C. to 40xc2x0 C., preferably of from 25xc2x0 C. to 40xc2x0 C. The body fluid may be directly reintroduced continuously or it may be collected and then be reintroduced. The method may be repeated as required prior to transplantation and/or after transplantation as long as removal of antibodies is of therapeutic benefit.
In accordance with the foregoing the present invention further provides:
(a) a conjugate Type II for use preferably as adsorbent in the affinity chromatography of body fluid;
(b) a method for removing antibodies from human body fluid, which method comprises extracorporally contacting said body fluid with a conjugate Type II as under (a) and reintroducing the body fluid into the patient""s body;
(c) a composition comprising a conjugate Type II as under (a) or a mixture of such conjugates Type II as hereinbefore disclosed for use preferably in a method for removing xenoantibodies from human body fluid; and
(d) an affinity chromatography cartridge comprising as adsorbent a conjugate Type II or a mixture therof or a composition as under (c).
Following examples illustrate the invention without limitation.
The following abbreviations are used in the examples: Ac: acetyl; Bn: benzyl; Bz: benzoyl; DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene; DMF: N,N-dimethyl formamide; DMTST: dimethylmethylthio-sulfonium trifluoromethylsulfonate; eq: equivalent(s); GlcNAc: N-acetylglucosamine; n: degree of polymerization; Sepharose 4 fast flow: NHS-activated Sepharose 4 Fast Flow (Pharmacia Biotech); TESOTf: triethylsilyl trifluoromethane-sulfonate; UDP-Gal: uridine-O(6)diphosphoryl-xcex1-D-galactose; RT: room temperature.
The mean molecular weights (M) and the polydispersities of the polylysine hydrobromides which are commercially obtainable from SIGMA are determined by means of viscosity measurements and SEC-LALLS (size exclusion chromatography/low angle laser light scattering) of the succinyl derivatives of the compounds.
A Preparation of Starting Compounds